The EMBO Journal vol.9 no.1 pp.251 -255, 1990
Discrete domains of human U6 snRNA required for the assembly of U4/U6 snRNP and splicing complexes
Albrecht Bindereif, Thorsten Wolff and Michael R.Green1 Max-Planck-Institut fur Molekulare Genetik, Otto-WarburgLaboratorium, Ihnestrasse 73, D-1000 Berlin 33 (Dahlem), FRG and 'Department of Biochemistry and Molecular Biology, Harvard University, 7 Divinity Avenue, Cambridge, MA 02138, USA Communicated by J.Tooze
U6 snRNA sequences required for assembly of U4/U6 snRNP and splicing complexes were determined by in vitro reconstitution of snRNPs. Both mutagenesis and chemical modification/interference assays identify a U6 snRNA domain required for U4/U6 snRNP formation. The results support the existence of a U4/U6 snRNA interaction domain previously proposed on the basis of phylogenetic evidence. In addition, two short U6 snRNA regions flanking the U4/U6 interaction domain are essential to assemble the U4/U6 snRNP into splicing complexes. These two regions may represent binding sites for splicing factors or may facilitate the formation of an alternative U6 snRNA secondary structure during spliceosome assembly. Key words: U4/U6 snRNP/snRNA/splicing
Introduction Pre-mRNA splicing occurs in a large complex, designated the spliceosome, which is composed of multiple small nuclear ribonucleoproteins (snRNPs) and proteins. Four snRNPs (U1, U2, U4/U6 and U5 snRNPs) are required for splicing and interact in an ordered fashion with the premRNA, each other and protein factors to form an active spliceosome (reviewed in Steitz et al., 1988). In the spliceosome, Ul and U2 snRNPs are tightly bound to the 5' splice site and branch site, respectively, rendering these regions resistant to RNase digestion. In contrast, U4/U6 and U5 snRNPs appear to interact primarily with Ul and U2 snRNPs and not with the pre-mRNA (Bindereif and Green, 1987). U6 snRNA has several unique properties (reviewed in Guthrie and Patterson, 1988). First, it is the most highly conserved spliceosomal snRNA. Second, U6 snRNA is the only spliceosomal snRNA that lacks the trimethylguanosine cap structure. Third, U4/U6 snRNP contains two snRNAs joined by inter-molecular base pairing. Since U6 snRNA lacks an Sm binding site, which is required for assembly with snRNP structural polypeptides, the assembly of U6 snRNA into a snRNP depends on association with U4 snRNP. The U4/U6 and U5 snRNPs interact to form a U4/U5/U6 multi-snRNP complex, which is apparently the form in which these snRNAs are incorporated into the spliceosome (Konarska and Sharp, 1987). Subsequently, the U4/U6 © Oxford University Press
snRNP undergoes a major conformational change as evidenced by the dissociation of U4 snRNA, but not of U6 snRNA from spliceosomal complexes resolved on nondenaturing gels (Pikielny et al., 1986; Cheng and Abelson, 1987; Lamond et al., 1988). This conformational change is concomitant with the appearance of splicing intermediates, suggesting a role in the catalytic activation of the spliceosome. We have recently described procedures for the in vitro reconstitution of a U4/U6 snRNP, which is assembled into a spliceosome (Pikielny et al., 1989). This allows us to delineate functional domains of U6 snRNA. Here we show that U6 snRNA contains at least two functional domains. One domain is required for formation of a U4/U6 snRNP, and the other domain enables the U4/U6 snRNP to be incorporated into the spliceosome.
Results and discussion Mapping the U4/U6 snRNP assembly domain of U6 snRNA To identify and map U6 snRNA regions essential for snRNP assembly, we reconstituted U4/U6 snRNPs using a series of U6 snRNA deletion derivatives (Figure 1). This in vitro reconstitution procedure used the endogenous U4 snRNP present in nuclear extract; under the reconstitution conditions, the added, 32P-labeled U6 snRNA was assembled into a U4/U6 snRNP. U4/U6 snRNP assembly was assayed by immunoprecipitation with anti-Sm antibodies (Pikielny et al., 1989). U6 snRNA alone does not form immunoprecipitable snRNP complexes; only in association with U4 snRNP can U6 snRNA be efficiently immunoprecipitated by anti-Sm antibodies (Bringmann et al., 1984; Hashimoto and Steitz, 1984). Figure IA and B shows that extensive deletions from both the 5' and 3' ends of U6 snRNA did not interfere with U4/U6 snRNP formation. U6 snRNAs deleted up to 52 nucleotides from the 5' end and 20 nucleotides from the 3' end, respectively, could still assemble to a U4/U6 snRNP with efficiencies >50% of wild-type SP6-U6 snRNA. Further 5' deletions prevented association with U4 snRNP. Further 3' deletions resulted in drastically decreased efficiencies (< 10%) of U4/U6 snRNP assembly (SP6-U6 3'A44, A48; Figure 1B) or abolished U4/U6 snRNP reconstitution completely (SP6-U6 3'A75; Figure iB). We conclude that an internal region of U6 snRNA is required for U4/U6 snRNP assembly. To identify this internal U6 snRNA region, we constructed and analyzed a series of U6 snRNA derivatives containing small internal deletions. Figure IC shows that three separate, contiguous U6 snRNA deletions between nucleotides 49 and 74 (SP6-U6AP28, SP6-U6AP20, SP6-U6AP1 1) completely inhibited U4/U6 snRNP formation. In contrast, a 10 nucleotide region further in the 3' direction was dispensable for
251
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S. Fig. 1. U4/U6 snRNP reconstitution of U6 snRNA derivatives. The 5'(A), 3'(B) and internal (C) deletions of SP6-U6 snRNA were reconstituted into U4/U6 snRNPs in nuclear extract. U4/U6 snRNP reconstitution was assayed by cs-Sm immunoprecipitation as described in Materials and methods. For each SP6-U6 snRNA derivative the total RNA after the reconstitution reaction in nuclear extract (Total RNA) and the cs-Sm immunoprecipitated RNA (cs-Sm) are shown.
snRNP assembly (nucleotides 75-84; SP6-U6AN7). It might be suprising that deleting most of the 3' half of U6 snRNA still allowed U4/U6 snRNP assembly, although at a very low efficiency (SP6-U6 3'A44; Figure IB), whereas deleting a minor internal part of this region gave no detectable snRNP formation (SP6-U6AP1 1; Figure IC). Possibly the change of adjacent U6 snRNA sequences in the internal deletion derivative causes this apparent discrepancy. In summary, deletion mapping identified a U6 snRNA region between nucleotides 53 and 74 that is essential for U4/U6 snRNP assembly. Next, we mapped the U6 snRNA nucleotides required for U4/U6 snRNP assembly by chemical modification/interference assays (Peattie, 1979; Conway and Wickens, 1987; Rymond and Rosbash, 1988). 3'-32P-end-labeled U6 snRNA was chemically modified with diethylpyrocarbonate (DEPC) or hydrazine. DEPC modifies purines by N7-carbethoxylation (under our conditions, adenosines reacted preferentially over guanosines), and hydrazine modifies pyrimidines (depending on the reaction conditions, resulting in base removal of uridines or cytosines). Modified U6 snRNA was then assembled in vitro into U4/U6 snRNP and immunoprecipitated from the reaction mixture with anti-
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Fig. 2. Chemical modification/interference analysis of U6 snRNA. The 3'-32P-end-labeled SP6-U6 snRNA is chemically modified by DEPC (carboxyethylation of purine bases: A>G) and by hydrazine (removal of pyrimidine bases: C or U). Modified SP6-U6 snRNA is then reconstituted in vitro into U4/U6 snRNP, which is purified by cs-Sm immunoprecipitation. The sites of modification are subsequently cleaved by aniline. Comparison of the cleavage pattern from total modified SP6-U6 snRNA and from modified SP6-U6 snRNA present in U4/U6 snRNP identifies U6 snRNA nucleotides that are essential in U4/U6 snRNP formation. For each of the modification reactions (A>G, C, U) is shown: RNA, total modified SP6-U6 snRNA before cleavage reaction; RNA (A>G, C, U), total modified SP6-U6 snRNA after cleavage reaction; NE-Total, total modified SP6-U6 snRNA after reconstitution and cleavage reaction; NE-U4/U6, modified SP6-U6 snRNA in U4/U6 snRNP after reconstitution, cs-Sm immunoprecipitation and cleavage reaction (interference); M, SP6-U6 partial alkaline hydrolysis ladder. The nucleotide positions of SP6-U6 snRNA where interference was detected are indicated on the left of each panel (see Figure 4 for a summary of these results).
Sm antibodies. RNA was purified from the immunoprecipitate and cleaved at the sites of modification with aniline. U6 snRNA interference patterns obtained with three different modification reactions are shown in Figure 2. Comparing the cleavage patterns obtained with total modified U6 snRNA after incubation in nuclear extract (lane NE-total) and with immunoprecipitated U6 snRNA (lane NE-U4/U6) identified nucleotides that are essential for U4/U6 snRNP formation. As controls, the cleavage patterns of modified U6 snRNA before reconstitution (lane RNA-A > G, -C, -U) and modified, uncleaved RNA (lane RNA) are shown. Figure 4 presents the positions of interference in the context of the proposed U4/U6 snRNA secondary structure. The region of interference is divided into two subregions of continuous strong interference, nucleotides 53-55 and 60-70, that are separated by four positions (nucleotides 56-59) of weak or no interference. In addition, weak
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Fig. 3. Spliceosome assembly with SP6-U6 snRNA derivatives (A)
32P-labeled wild-type SP6-U6 snRNA was reconstituted in vitro to U4/U6 snRNP and added to a splicing reaction containing unlabeled pre-mRNA (SPAd/BgII) or a non-specific RNA (Gem3/AsuI) as described in Materials and methods. For the ATP control (lane ATP -/+), 100 ng pre-mRNA was used per 1/2 X-reaction. For the specificity control (lane NS), 100 ng Gem3/AsuI RNA were used. To obtain marker positions for the A and B complexes, 32P-labeled SPAd/Bgll pre-mRNA was incubated under splicing conditions (lane M; positions of A and B complexes indicated on the sides of the panels). (B) 32P-labeled wild-type SP6-U6 snRNA and SP6-U6 snRNA derivatives were reconstituted in vitro to U4/U6 snRNPs and added to a splicing reaction containing unlabeled pre-mRNA (MINX/BamHI; 100 ng per 25 1l reaction) as described in Materials and methods (premRNA +). Assembly of U6 snRNAs into splicing complexes after the indicated times of incubation (20 min, 40 min) was monitored by the appearance of 32P-labeled U6 snRNA in the B complex as analyzed by native RNP gel electrophoresis. To obtain marker positions for the A and B complexes, 32P-labeled MINX/BamHI pre-mRNA was incubated under splicing conditions (lane M; positions of A and B complexes indicated on the left of each panel).
interference was detected at three nucleotides located on each side of the U4/U6 snRNP assembly region (nucleotides 50-52 and 71-73). Placement of the 5' boundary of the U4/U6 snRNP assembly region differed slightly between the chemical modification/interference and the deletion analysis: chemical modification of the three nucleotides at the 5' end of stem I in U6 snRNA (nucleotides 50-52) weakly interfered with U4/U6 snRNP assembly, whereas deleting this region as part of a large 5' deletion (SP6-U6 5'A52) had no effect. We believe that chemical modification/interference is a more sensitive assay for detecting subtle effects of mutations; in the interference experiments, a large number of differentially
modified U6 snRNAs compete with each other for association with U4 snRNP. In contrast, in deletion analysis a relatively large quantity of a single U6 snRNA derivative is tested. In recent phylogenetic studies a U6 snRNA secondary structure was proposed, in which U4 and U6 snRNAs extensively interact by base pairing (Brow and Guthrie, 1988; Guthrie and Patterson, 1988; Zucker-Aprison et al., 1988). Two U4/U6 intermolecular helices and a U4 intramolecular stem-loop structure are organized in a 'Y structure' (see Figure 4). Part of the U4 - U6 snRNA basepairing interaction had previously been detected by psoralen crosslinking of U4 and U6 snRNAs in the U4/U6 snRNP (Rinke etal., 1985). Our deletion and interference analysis provides strong experimental evidence for this phylogenetic model. Most likely, the U4/U6 snRNA interaction region functions as a discrete U4/U6 snRNP assembly domain. It is striking that within stem I and II, single-base modifications of both A -U and G-C base pairs dramatically affected U4/U6 snRNP assembly. There is also one position within stem II (nucleotide C62), which is not involved in the proposed base pairing, yet exhibited a strong interference effect. In addition, modification at nucleotide A73, which is outside of stem II, weakly interfered. One interpretation of these results is that U4/U6 snRNP assembly may involve RNA -protein interactions in addition to RNA -RNA base pairing. The putative protein could be either one of the known snRNP polypeptides (for review, see Liihrmann, 1988) or an as yet unidentified U4/U6 snRNP-specific protein. Identifying spliceosome assembly domains in U6 snRNA We next asked whether there are U6 snRNA regions in addition to the U4/U6 snRNP assembly domain required for assembly of U6 snRNA into splicing complexes. We tested the ability of the U6 snRNA derivatives described above to be incorporated into splicing complexes. 32P-labeled U6 snRNA derivatives were assembled into U4/U6 snRNPs and added to a splicing reaction containing an unlabeled premRNA substrate. Incorporation of the 32P-labeled U4/U6 snRNP complexes into splicing complexes was analyzed by native gel electrophoresis. This gel system resolves two complexes from each other (Konarska and Sharp, 1986): A (U2 snRNP) and B (U2, U4/U6, U5 snRNPs) (see Figure 3, marker lanes). The appearance of [32P]U6 snRNA-label in the B complex is evidence for a U6 snRNA derivative being functional in splicing complex assembly. As expected, wild-type 32P-labeled U6 snRNA was absent from the A complex and was assembled into the B complex (Figure 3). Using several criteria we confirmed that this complex actually represents authentic splicing complex (Figure 3A and Pikielny et al., 1989). First, it comigrates with 32P-pre-mRNA-labeled B complex, and its formation is ATP-dependent. Second, its formation requires the addition of pre-mRNA splicing substrate and depends on the concentration of added splicing substrate. Third, it is specific for a pre-mRNA splicing substrate (Figure 3A). Likewise a U6 snRNA lacking 37 nucleotides from the 5' end was efficiently assembled into a B complex (SP6-U6 5'A37; Figure 3B); a U6 snRNA deletion of 20 nucleotides from the 3' end also did not affect assembly into splicing complexes (SP6-U6 3'A20; Figure 3B). In contrast, deleting
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Fig. 4. snRNP and spliceosome assembly domains of U6 snRNA and secondary structure model of human U4 and U6 snRNAs. The SP6-U6 snRNA derivatives used in this study are schematically represented (deleted regions indicated by thin lines). To the right, the ability of these SP6-U6 snRNA derivatives to assemble into U4/U6 snRNPs and into splicing complexes is indicated [+ indicates efficiencies of U4/U6 snRNP and spliceosome assembly of >50% of that of wild-type SP6-U6 snRNA; +/- and - indicate corresponding efficiencies of < 10% and undetectable levels, respectively (see Figure 1 and 3B, and data not shown)]. Below, the boundaries of the U4/U6 snRNP assembly domain of U6 snRNA are outlined as well as the boundaries to which the spliceosome assembly domains maximally extend. On the lower half, the proposed secondary structure of human U4 and U6 snRNAs is shown (corresponding to the proposed consensus U4/U6 snRNA secondary structure; Guthrie and Patterson, 1988). The boundaries of the SP6-U6 snRNA derivatives as well as the Sm binding site of U4 snRNA (boxed sequence) are also indicated. Under the U6 snRNA sequence, the results of U6 snRNA interference assays are schematically represented (see Figure 2; interference of U4/U6 snRNP assembly: no interference, open circles; weak interference, half-closed circles; strong interference, closed circles).
52 nucleotides from the 5' end abolished the ability of U6 snRNA to be incorporated into a splicing complex (SP6-U6 5'A52: Figure 3B). Similarly, U6 snRNA with a small internal deletion, SP6-U6AN7, was practically inactive in splicing complex assembly (efficiency
Discrete domains of human U6 snRNA required for the assembly of U4/U6 snRNP and splicing complexes
Albrecht Bindereif, Thorsten Wolff and Michael R.Green1 Max-Planck-Institut fur Molekulare Genetik, Otto-WarburgLaboratorium, Ihnestrasse 73, D-1000 Berlin 33 (Dahlem), FRG and 'Department of Biochemistry and Molecular Biology, Harvard University, 7 Divinity Avenue, Cambridge, MA 02138, USA Communicated by J.Tooze
U6 snRNA sequences required for assembly of U4/U6 snRNP and splicing complexes were determined by in vitro reconstitution of snRNPs. Both mutagenesis and chemical modification/interference assays identify a U6 snRNA domain required for U4/U6 snRNP formation. The results support the existence of a U4/U6 snRNA interaction domain previously proposed on the basis of phylogenetic evidence. In addition, two short U6 snRNA regions flanking the U4/U6 interaction domain are essential to assemble the U4/U6 snRNP into splicing complexes. These two regions may represent binding sites for splicing factors or may facilitate the formation of an alternative U6 snRNA secondary structure during spliceosome assembly. Key words: U4/U6 snRNP/snRNA/splicing
Introduction Pre-mRNA splicing occurs in a large complex, designated the spliceosome, which is composed of multiple small nuclear ribonucleoproteins (snRNPs) and proteins. Four snRNPs (U1, U2, U4/U6 and U5 snRNPs) are required for splicing and interact in an ordered fashion with the premRNA, each other and protein factors to form an active spliceosome (reviewed in Steitz et al., 1988). In the spliceosome, Ul and U2 snRNPs are tightly bound to the 5' splice site and branch site, respectively, rendering these regions resistant to RNase digestion. In contrast, U4/U6 and U5 snRNPs appear to interact primarily with Ul and U2 snRNPs and not with the pre-mRNA (Bindereif and Green, 1987). U6 snRNA has several unique properties (reviewed in Guthrie and Patterson, 1988). First, it is the most highly conserved spliceosomal snRNA. Second, U6 snRNA is the only spliceosomal snRNA that lacks the trimethylguanosine cap structure. Third, U4/U6 snRNP contains two snRNAs joined by inter-molecular base pairing. Since U6 snRNA lacks an Sm binding site, which is required for assembly with snRNP structural polypeptides, the assembly of U6 snRNA into a snRNP depends on association with U4 snRNP. The U4/U6 and U5 snRNPs interact to form a U4/U5/U6 multi-snRNP complex, which is apparently the form in which these snRNAs are incorporated into the spliceosome (Konarska and Sharp, 1987). Subsequently, the U4/U6 © Oxford University Press
snRNP undergoes a major conformational change as evidenced by the dissociation of U4 snRNA, but not of U6 snRNA from spliceosomal complexes resolved on nondenaturing gels (Pikielny et al., 1986; Cheng and Abelson, 1987; Lamond et al., 1988). This conformational change is concomitant with the appearance of splicing intermediates, suggesting a role in the catalytic activation of the spliceosome. We have recently described procedures for the in vitro reconstitution of a U4/U6 snRNP, which is assembled into a spliceosome (Pikielny et al., 1989). This allows us to delineate functional domains of U6 snRNA. Here we show that U6 snRNA contains at least two functional domains. One domain is required for formation of a U4/U6 snRNP, and the other domain enables the U4/U6 snRNP to be incorporated into the spliceosome.
Results and discussion Mapping the U4/U6 snRNP assembly domain of U6 snRNA To identify and map U6 snRNA regions essential for snRNP assembly, we reconstituted U4/U6 snRNPs using a series of U6 snRNA deletion derivatives (Figure 1). This in vitro reconstitution procedure used the endogenous U4 snRNP present in nuclear extract; under the reconstitution conditions, the added, 32P-labeled U6 snRNA was assembled into a U4/U6 snRNP. U4/U6 snRNP assembly was assayed by immunoprecipitation with anti-Sm antibodies (Pikielny et al., 1989). U6 snRNA alone does not form immunoprecipitable snRNP complexes; only in association with U4 snRNP can U6 snRNA be efficiently immunoprecipitated by anti-Sm antibodies (Bringmann et al., 1984; Hashimoto and Steitz, 1984). Figure IA and B shows that extensive deletions from both the 5' and 3' ends of U6 snRNA did not interfere with U4/U6 snRNP formation. U6 snRNAs deleted up to 52 nucleotides from the 5' end and 20 nucleotides from the 3' end, respectively, could still assemble to a U4/U6 snRNP with efficiencies >50% of wild-type SP6-U6 snRNA. Further 5' deletions prevented association with U4 snRNP. Further 3' deletions resulted in drastically decreased efficiencies (< 10%) of U4/U6 snRNP assembly (SP6-U6 3'A44, A48; Figure 1B) or abolished U4/U6 snRNP reconstitution completely (SP6-U6 3'A75; Figure iB). We conclude that an internal region of U6 snRNA is required for U4/U6 snRNP assembly. To identify this internal U6 snRNA region, we constructed and analyzed a series of U6 snRNA derivatives containing small internal deletions. Figure IC shows that three separate, contiguous U6 snRNA deletions between nucleotides 49 and 74 (SP6-U6AP28, SP6-U6AP20, SP6-U6AP1 1) completely inhibited U4/U6 snRNP formation. In contrast, a 10 nucleotide region further in the 3' direction was dispensable for
251
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S. Fig. 1. U4/U6 snRNP reconstitution of U6 snRNA derivatives. The 5'(A), 3'(B) and internal (C) deletions of SP6-U6 snRNA were reconstituted into U4/U6 snRNPs in nuclear extract. U4/U6 snRNP reconstitution was assayed by cs-Sm immunoprecipitation as described in Materials and methods. For each SP6-U6 snRNA derivative the total RNA after the reconstitution reaction in nuclear extract (Total RNA) and the cs-Sm immunoprecipitated RNA (cs-Sm) are shown.
snRNP assembly (nucleotides 75-84; SP6-U6AN7). It might be suprising that deleting most of the 3' half of U6 snRNA still allowed U4/U6 snRNP assembly, although at a very low efficiency (SP6-U6 3'A44; Figure IB), whereas deleting a minor internal part of this region gave no detectable snRNP formation (SP6-U6AP1 1; Figure IC). Possibly the change of adjacent U6 snRNA sequences in the internal deletion derivative causes this apparent discrepancy. In summary, deletion mapping identified a U6 snRNA region between nucleotides 53 and 74 that is essential for U4/U6 snRNP assembly. Next, we mapped the U6 snRNA nucleotides required for U4/U6 snRNP assembly by chemical modification/interference assays (Peattie, 1979; Conway and Wickens, 1987; Rymond and Rosbash, 1988). 3'-32P-end-labeled U6 snRNA was chemically modified with diethylpyrocarbonate (DEPC) or hydrazine. DEPC modifies purines by N7-carbethoxylation (under our conditions, adenosines reacted preferentially over guanosines), and hydrazine modifies pyrimidines (depending on the reaction conditions, resulting in base removal of uridines or cytosines). Modified U6 snRNA was then assembled in vitro into U4/U6 snRNP and immunoprecipitated from the reaction mixture with anti-
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Fig. 2. Chemical modification/interference analysis of U6 snRNA. The 3'-32P-end-labeled SP6-U6 snRNA is chemically modified by DEPC (carboxyethylation of purine bases: A>G) and by hydrazine (removal of pyrimidine bases: C or U). Modified SP6-U6 snRNA is then reconstituted in vitro into U4/U6 snRNP, which is purified by cs-Sm immunoprecipitation. The sites of modification are subsequently cleaved by aniline. Comparison of the cleavage pattern from total modified SP6-U6 snRNA and from modified SP6-U6 snRNA present in U4/U6 snRNP identifies U6 snRNA nucleotides that are essential in U4/U6 snRNP formation. For each of the modification reactions (A>G, C, U) is shown: RNA, total modified SP6-U6 snRNA before cleavage reaction; RNA (A>G, C, U), total modified SP6-U6 snRNA after cleavage reaction; NE-Total, total modified SP6-U6 snRNA after reconstitution and cleavage reaction; NE-U4/U6, modified SP6-U6 snRNA in U4/U6 snRNP after reconstitution, cs-Sm immunoprecipitation and cleavage reaction (interference); M, SP6-U6 partial alkaline hydrolysis ladder. The nucleotide positions of SP6-U6 snRNA where interference was detected are indicated on the left of each panel (see Figure 4 for a summary of these results).
Sm antibodies. RNA was purified from the immunoprecipitate and cleaved at the sites of modification with aniline. U6 snRNA interference patterns obtained with three different modification reactions are shown in Figure 2. Comparing the cleavage patterns obtained with total modified U6 snRNA after incubation in nuclear extract (lane NE-total) and with immunoprecipitated U6 snRNA (lane NE-U4/U6) identified nucleotides that are essential for U4/U6 snRNP formation. As controls, the cleavage patterns of modified U6 snRNA before reconstitution (lane RNA-A > G, -C, -U) and modified, uncleaved RNA (lane RNA) are shown. Figure 4 presents the positions of interference in the context of the proposed U4/U6 snRNA secondary structure. The region of interference is divided into two subregions of continuous strong interference, nucleotides 53-55 and 60-70, that are separated by four positions (nucleotides 56-59) of weak or no interference. In addition, weak
Assembly of U4/U6 snRNP
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Fig. 3. Spliceosome assembly with SP6-U6 snRNA derivatives (A)
32P-labeled wild-type SP6-U6 snRNA was reconstituted in vitro to U4/U6 snRNP and added to a splicing reaction containing unlabeled pre-mRNA (SPAd/BgII) or a non-specific RNA (Gem3/AsuI) as described in Materials and methods. For the ATP control (lane ATP -/+), 100 ng pre-mRNA was used per 1/2 X-reaction. For the specificity control (lane NS), 100 ng Gem3/AsuI RNA were used. To obtain marker positions for the A and B complexes, 32P-labeled SPAd/Bgll pre-mRNA was incubated under splicing conditions (lane M; positions of A and B complexes indicated on the sides of the panels). (B) 32P-labeled wild-type SP6-U6 snRNA and SP6-U6 snRNA derivatives were reconstituted in vitro to U4/U6 snRNPs and added to a splicing reaction containing unlabeled pre-mRNA (MINX/BamHI; 100 ng per 25 1l reaction) as described in Materials and methods (premRNA +). Assembly of U6 snRNAs into splicing complexes after the indicated times of incubation (20 min, 40 min) was monitored by the appearance of 32P-labeled U6 snRNA in the B complex as analyzed by native RNP gel electrophoresis. To obtain marker positions for the A and B complexes, 32P-labeled MINX/BamHI pre-mRNA was incubated under splicing conditions (lane M; positions of A and B complexes indicated on the left of each panel).
interference was detected at three nucleotides located on each side of the U4/U6 snRNP assembly region (nucleotides 50-52 and 71-73). Placement of the 5' boundary of the U4/U6 snRNP assembly region differed slightly between the chemical modification/interference and the deletion analysis: chemical modification of the three nucleotides at the 5' end of stem I in U6 snRNA (nucleotides 50-52) weakly interfered with U4/U6 snRNP assembly, whereas deleting this region as part of a large 5' deletion (SP6-U6 5'A52) had no effect. We believe that chemical modification/interference is a more sensitive assay for detecting subtle effects of mutations; in the interference experiments, a large number of differentially
modified U6 snRNAs compete with each other for association with U4 snRNP. In contrast, in deletion analysis a relatively large quantity of a single U6 snRNA derivative is tested. In recent phylogenetic studies a U6 snRNA secondary structure was proposed, in which U4 and U6 snRNAs extensively interact by base pairing (Brow and Guthrie, 1988; Guthrie and Patterson, 1988; Zucker-Aprison et al., 1988). Two U4/U6 intermolecular helices and a U4 intramolecular stem-loop structure are organized in a 'Y structure' (see Figure 4). Part of the U4 - U6 snRNA basepairing interaction had previously been detected by psoralen crosslinking of U4 and U6 snRNAs in the U4/U6 snRNP (Rinke etal., 1985). Our deletion and interference analysis provides strong experimental evidence for this phylogenetic model. Most likely, the U4/U6 snRNA interaction region functions as a discrete U4/U6 snRNP assembly domain. It is striking that within stem I and II, single-base modifications of both A -U and G-C base pairs dramatically affected U4/U6 snRNP assembly. There is also one position within stem II (nucleotide C62), which is not involved in the proposed base pairing, yet exhibited a strong interference effect. In addition, modification at nucleotide A73, which is outside of stem II, weakly interfered. One interpretation of these results is that U4/U6 snRNP assembly may involve RNA -protein interactions in addition to RNA -RNA base pairing. The putative protein could be either one of the known snRNP polypeptides (for review, see Liihrmann, 1988) or an as yet unidentified U4/U6 snRNP-specific protein. Identifying spliceosome assembly domains in U6 snRNA We next asked whether there are U6 snRNA regions in addition to the U4/U6 snRNP assembly domain required for assembly of U6 snRNA into splicing complexes. We tested the ability of the U6 snRNA derivatives described above to be incorporated into splicing complexes. 32P-labeled U6 snRNA derivatives were assembled into U4/U6 snRNPs and added to a splicing reaction containing an unlabeled premRNA substrate. Incorporation of the 32P-labeled U4/U6 snRNP complexes into splicing complexes was analyzed by native gel electrophoresis. This gel system resolves two complexes from each other (Konarska and Sharp, 1986): A (U2 snRNP) and B (U2, U4/U6, U5 snRNPs) (see Figure 3, marker lanes). The appearance of [32P]U6 snRNA-label in the B complex is evidence for a U6 snRNA derivative being functional in splicing complex assembly. As expected, wild-type 32P-labeled U6 snRNA was absent from the A complex and was assembled into the B complex (Figure 3). Using several criteria we confirmed that this complex actually represents authentic splicing complex (Figure 3A and Pikielny et al., 1989). First, it comigrates with 32P-pre-mRNA-labeled B complex, and its formation is ATP-dependent. Second, its formation requires the addition of pre-mRNA splicing substrate and depends on the concentration of added splicing substrate. Third, it is specific for a pre-mRNA splicing substrate (Figure 3A). Likewise a U6 snRNA lacking 37 nucleotides from the 5' end was efficiently assembled into a B complex (SP6-U6 5'A37; Figure 3B); a U6 snRNA deletion of 20 nucleotides from the 3' end also did not affect assembly into splicing complexes (SP6-U6 3'A20; Figure 3B). In contrast, deleting
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A.Bindereif, T.Wolff and M.R.Green Assembly of
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Fig. 4. snRNP and spliceosome assembly domains of U6 snRNA and secondary structure model of human U4 and U6 snRNAs. The SP6-U6 snRNA derivatives used in this study are schematically represented (deleted regions indicated by thin lines). To the right, the ability of these SP6-U6 snRNA derivatives to assemble into U4/U6 snRNPs and into splicing complexes is indicated [+ indicates efficiencies of U4/U6 snRNP and spliceosome assembly of >50% of that of wild-type SP6-U6 snRNA; +/- and - indicate corresponding efficiencies of < 10% and undetectable levels, respectively (see Figure 1 and 3B, and data not shown)]. Below, the boundaries of the U4/U6 snRNP assembly domain of U6 snRNA are outlined as well as the boundaries to which the spliceosome assembly domains maximally extend. On the lower half, the proposed secondary structure of human U4 and U6 snRNAs is shown (corresponding to the proposed consensus U4/U6 snRNA secondary structure; Guthrie and Patterson, 1988). The boundaries of the SP6-U6 snRNA derivatives as well as the Sm binding site of U4 snRNA (boxed sequence) are also indicated. Under the U6 snRNA sequence, the results of U6 snRNA interference assays are schematically represented (see Figure 2; interference of U4/U6 snRNP assembly: no interference, open circles; weak interference, half-closed circles; strong interference, closed circles).
52 nucleotides from the 5' end abolished the ability of U6 snRNA to be incorporated into a splicing complex (SP6-U6 5'A52: Figure 3B). Similarly, U6 snRNA with a small internal deletion, SP6-U6AN7, was practically inactive in splicing complex assembly (efficiency
